Cell Line Processing (Tissue culture and freezing to save DNA for cloning)

Obtaining a Tissue Sample for Cell Culture and Freezing

Cells should be collected and placed directly into cold culture
medium (DMEM/F-12 with 10% fetal bovine serum and antibiotics). We
can send the medium; it is sent chilled in three 15-ml conical
tubes. Alternatively, chilled embryo holding medium may be used;
using sterile technique, fill three 5- to 15-ml sterile sealable
tubes 3⁄4 full with medium.

The tubes should be refrigerated until use. They should be
placed in an insulated (e.g. Styrofoam) container (with cover), on
ice, when going to take the tissue sample.

Before going to take the sample, make sure you have something
to label the tubes with, and that there is someone ready to open
the tubes, who has a 1.5 inch 18- or 20-ga needle ready to help get
the tissue off the forceps.

We typically take tissue from the neck under the mane for
cosmetic reasons and because this may have had less sun exposure.
For an animal that may be terminal, it is recommended that two
different areas be sampled, and the tissue grown as two separate
cell lines. In this case take another sample from the gum if
possible, or if not, from any other place easy to get to, which has
been out of the sun. Try to avoid really fatty areas such as the
tail head.

Shave an area approximately 7 x 7 cm (unless gum), and scrub
and rinse as for surgery. We typically use betadine scrub rather
than nolvasan. Do a last wipe with only sterile saline to remove
remnants of scrub.

Glove and maintain sterility; contamination is the most common
problem with tissue biopsies.

Perform an inverted U block with lidocaine, so that the
lidocaine is not in the tissue being sampled.

Make a small skin incision (about 2 cm), and evert the skin
edges. Obtain small (~5 mm3) samples of subcutaneous connective
tissue and place them immediately into the cooled cell culture
medium. Place 1 or 2 pieces of tissue in each tube. The assistant,
using the needle, should help move the tissue quickly, and should
make sure the tissue goes down into the medium. If you think any
contamination may have taken place, start with a new tube, and new
instruments if necessary.

If taking samples from two areas, put the samples in different
tubes and label accordingly.

Keep the medium and samples cold, and cover the container so
the tubes are not exposed to sun or fluorescent light. Transport as
quickly as possible to the laboratory. Shipping address: Dr. Katrin
Hinrichs, Large Animal Front Desk, Texas A&M University College
of Veterinary Medicine, College Station, TX 77843-4457.

Close incision with a couple of sutures.

If you have questions regarding the tissue collection or
processing, please contact: